Expression and purification of His-tagged proteins were performed as described23 (link),24 (link),26 (link). E. Coli BL21-Gold (DE3) Competent Cells (Agilent Technologies) were transformed with the plasmid coding for the different proteins. Cells were grown at 37 °C in 2YT medium containing 200 µg.L−1 ampicillin until the OD600nm reached 0.6. Protein expression was induced by addition of 1 mM IPTG and the cells were further incubated for 5 h at 37 °C. The cells were harvested, suspended in 50 mM sodium phosphate pH7.5, 150 mM NaCl (PBS), submitted to three freezing/thawing cycles, treated with DNase I for 30 min and sonicated. The His-tagged proteins were purified using nickel-affinity chromatography (Histrap™ FF crude 5 mL, GE Healthcare). The His-tag A3-A3 and A3-bGFPD was cleaved by TEV protease for some SPR experiments.
Twin-Strep®-tagged proteins (bA3-2, eGFP and BFP) were also purified using a StrepTactin Sepharose affinity chromatography (StrepTrap™ HP 5 mL, GE Healthcare); both samples obtained from this first step purification were then submitted to a size exclusion chromatography.
Twin-Strep®-tagged proteins (bA3-2, eGFP and BFP) were also purified using a StrepTactin Sepharose affinity chromatography (StrepTrap™ HP 5 mL, GE Healthcare); both samples obtained from this first step purification were then submitted to a size exclusion chromatography.
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